miR-17-5p靶向乙醛脱氢酶2减少肺炎链球菌诱导的人肺腺癌肺泡基底上皮细胞系A549损伤

基础医学与临床 ›› 2020, Vol. 40 ›› Issue (10): 1362-1368.

miR-17-5p靶向乙醛脱氢酶2减少肺炎链球菌诱导的人肺腺癌肺泡基底上皮细胞系A549损伤

李维维¹, 葛斌², 邓涛^1*, 唐建军¹, 骆财元¹, 冯圣芳¹, 毛羽¹

1.成都市郫都区人民医院儿科;
2.检验科, 四川成都 611730

收稿日期:2019-08-26 修回日期:2020-01-02 出版日期:2020-10-05 发布日期:2020-09-29
通讯作者: ^* 124098403@qq.com
基金资助:
四川省医学会科研课题(S16050)

miR-17-5p targeting ALDH2 reduces the damage of human lung adenocarcinoma alveolar basal epithelial tissue cell line A549 induced by Streptococcus pneumoniae

LI Wei-wei¹, GE Bin², DENG Tao^1*, TANG Jian-jun¹, LUO Cai-yuan¹, FENG Sheng-fang¹, MAO Yu¹

1. Department of Pediatrics;
2. Department of Clinical Laboratory,Pidu District People's Hospital, Chengdu 611730, China

Received:2019-08-26 Revised:2020-01-02 Online:2020-10-05 Published:2020-09-29
Contact: ^* 124098403@qq.com

摘要/Abstract

摘要： 目的探讨miR-17-5p表达对肺炎链球菌(S.pneumoniae)诱导的人肺腺癌肺泡基底上皮细胞系A549损伤的影响及其作用机制。方法将A549细胞分为对照组、感染组(1×10⁸ CFU/mL S.pneumoniae);anti-miR-17-5p干预组(S.pneumoniae感染前转染anti-miR-17-5p)、anti-miR-NC干预组(S.pneumoniae感染前转染anti-miR-NC)、pcDNA-乙醛脱氢酶2(ALDH2)干预组(S.pneumoniae感染前转染pcDNA-ALDH2)、pcDNA干预组(S.pneumoniae感染前转染pcDNA)。RT-qPCR检测细胞中miR-17-5p与ALDH2 mRNA水平,酶联免疫吸附剂法检测细胞培养上清中IL-6和IL-10水平,流式细胞计量术检测细胞凋亡,Western blot检测ALDH2、Bcl-2和Bax蛋白水平。双荧光素酶实验验证miR-17-5p与ALDH2之间靶向关系。结果与对照组比,感染组miR-17-5p水平显著升高(P＜0.05);ALDH2 mRNA和蛋白水平显著降低(P＜0.05);IL-10和Bcl-2水平显著降低(P＜0.05);凋亡率、IL-6和Bax水平显著升高(P＜0.05)。与anti-miR-NC干预组比,anti-miR-17-5p干预组IL-10和Bcl-2水平显著升高(P＜0.05);凋亡率、IL-6和Bax水平显著降低(P＜0.05)。与pcDNA干预组比,pcDNA-ALDH2干预组IL-10和Bcl-2水平显著升高(P＜0.05);凋亡率、IL-6和Bax水平显著降低(P＜0.05)。miR-17-5p负调控ALDH2表达,抑制ALDH2表达逆转了抑制miR-17-5p表达对S.pneumoniae诱导的A549细胞损伤。结论抑制miR-17-5p表达通过调控ALDH2降低S.pneumoniae诱导的人肺腺癌肺泡基底上皮细胞系A549损伤。

关键词: miR-17-5p, 乙醛脱氢酶2, 肺炎链球菌, 肺泡上皮细胞, 细胞损伤

Abstract: Objective To investigate the effect of miR-17-5p expression on the injury of human lung adenocarcinoma alveolar basal epithelial cell line A549 induced by Streptococcus pneumonia and its mechanism. Methods A549 cells was divided into control group,infected group(1×10⁸ CFU/mL S.pneumoniae),anti-miR-17-5p intervention group (transfected with anti-miR-17-5p before S.pneumoniae infection),anti-miR-NC intervention group (transfected with anti-miR-NC before S.pneumoniae infection), and pcDNA-ALDH2 intervention group(trans- fected with pcDNA-ALDH2 before S.pneumoniae infection),and pcDNA intervention group (transfected with pcDNA before S.pneumoniae infection).The level of miR-17-5p and ALDH2 mRNA was detected by RT-qPCR. IL-6 and IL-10 in cell culture supernatant were detected by enzyme-linked immunosorbent assay. Apoptosis was detected by flow cytometry. ALDH2, Bcl-2 and Bax protein were detected by Western blot. Dual luciferase assay verified the targeting relationship between miR-17-5p and ALDH2. Results Compared with the control group, the level of miR-17-5p in the infected group was significantly increased (P＜0.05), while mRNA and the protein concentration of ALDH2, IL-10 and Bcl-2 were significantly decreased (P＜0.05). However, apoptotic rate and the levels of IL-6 and Bax were significantly increased (P＜0.05). Compared with the infected+anti-miR-NC group, IL-10 and Bcl-2 in the infected+anti-miR-17-5p group were significantly increased(P＜0.05), the apoptotic rate, IL-6 and Bax were significantly decreased (P＜0.05). Compared with the infected+pcDNA group, the levels of IL-10 and Bcl-2 in the infected+pcDNA-ALDH2 group were significantly increased (P＜0.05), and the apoptotic rate and the levels of IL-6 and Bax were significantly decreased (P＜0.05).miR-17-5p negatively regulated ALDH2 expression.Inhibition of ALDH2 expression reversed the effect of inhibiting miR-17-5p expression on S.pneumoniae-induced A549 cells damage. Conclusions Inhibition of miR-17-5p expression reduces S.pneumoniae-induced the damage of the cell line A549 by regulating ALDH2.

Key words: miR-17-5p, Aldehyde dehydrogenase 2(ALDH2), Streptococcus pneumonia, alveolar epithelial cells, cell injury

中图分类号:

R378

李维维, 葛斌, 邓涛, 唐建军, 骆财元, 冯圣芳, 毛羽. miR-17-5p靶向乙醛脱氢酶2减少肺炎链球菌诱导的人肺腺癌肺泡基底上皮细胞系A549损伤[J]. 基础医学与临床, 2020, 40(10): 1362-1368.

LI Wei-wei, GE Bin, DENG Tao, TANG Jian-jun, LUO Cai-yuan, FENG Sheng-fang, MAO Yu. miR-17-5p targeting ALDH2 reduces the damage of human lung adenocarcinoma alveolar basal epithelial tissue cell line A549 induced by Streptococcus pneumoniae[J]. Basic & Clinical Medicine, 2020, 40(10): 1362-1368.

参考文献

[1] 林琳, 刘晓民, 贾琰琰, 等. 关于肺炎链球菌易感人群的研究进展[J]. 临床肺科杂志, 2016, 21:1110-1112.
[2] 顾超, 刘元顺, 吕云, 等. Ⅱ型肺泡上皮细胞凋亡在肺气肿大鼠肺功能下降中的作用研究[J]. 浙江临床医学, 2017, 19:1585-1587.
[3] Shen Z, Zhou Y, Qu L, et al. ATP serves an anti-inflammatory role by enhancing β-defensin-2 response in acute pneumonia of rat[J]. Biomed Rep, 2017, 6:649-653.
[4] 刘红兵, 王凤斌, 张皓云, 等. 转染miR-17-5p对PC12细胞Bcl-2、Bax蛋白表达和形态学影响[J]. 神经解剖学杂志, 2016, 32:477-481.
[5] 王雷敏. mir-17在H₂O₂诱导的心肌细胞凋亡中的作用[D]. 哈尔滨,哈尔滨医科大学, 2014:1-3.
[6] 徐洪涛, 余健, 聂国明, 等. ALDH2在高氧引起的肺泡上皮细胞损伤中的作用[J]. 华南国防医学杂志, 2014, 28:34-37.
[7] 王勇, 孙朝峰, 胡钱红. NF-κB p65对肺炎链球菌诱导的肺泡上皮细胞凋亡及炎性因子表达的影响[J]. 实用预防医学, 2018, 25: 547-550.
[8] 吕永智, 雍康. 呼吸系统上皮细胞与巨噬细胞miRNA在哺乳动物肺炎发病机制中的作用[J]. 中国兽医杂志, 2017, 53:71-73.
[9] 郑颖, 李强, 阳慧, 等. 冠状动脉严重狭窄患者血浆miRNA-195表达及其与侧支循环形成的关系[J]. 实用医学杂志, 2018, 34:265-268.
[10] Feng A, Yuan X, Li X. miR-345 inhibits metasta-sis and epithelial-mesenchymal transition of gastric cancer by targeting FOXQ1[J]. Oncol Rep, 2017, 38:2752-2760.
[11] 于冰冰, 王素梅, 彭丹晖, 等. 不同浓度CO₂对肺泡Ⅱ型上皮细胞模型A549细胞活性的影响[J]. 广西医学, 2015, 37:150-153.
[12] 穆克利, 张蕾, 曹远国, 等. 医院获得性肺炎患者炎症因子的相关性研究[J]. 中华医院感染学杂志, 2017, 27:770-773.
[13] 刁云锋, 张士俊, 赵万勇, 等. 血必净注射液对重症肺炎患者血浆IL-6和TNF-α水平的影响[J]. 中草药, 2017, 48:1188-1191.
[14] 张俊波, 印双红, 张红. TLR 4在绵羊肺炎支原体介导肺泡上皮细胞凋亡中的作用机理[J]. 中国兽医杂志, 2017, 53:3-7.
[15] 杨丽, 张俊峰, 杨文龙, 等. ALDH2对大鼠心肌缺血/再灌注损伤与凋亡的影响及机制的研究[J]. 中国循证心血管医学杂志, 2018, 10:82-86.